Covalent Phosphoenzyme Research Articles

Reaction mechanism of Ca2+ ATPase of sarcoplasmic reticulum. Equilibrium and transient study of phosphorylation with Ca.ATP as substrate.

At pH 7.0 and 5 degrees C, in the absence of potassium and magnesium, the Ca-ATPase of the sarcoplasmic reticulum slowly hydrolyzes the Ca.ATP at a rate of 0.05 s-1. During turnover 4 nmol of phosphoenzyme per mg of total protein accumulate with a Km value of 10(-8) M. Combining rapid filtration and rapid acid quenching, we took advantage of the above properties to study the early steps of phosphorylation. (formula; see text) Direct measurements by rapid filtration of the rate constant of Ca.ATP binding (k1 = 3.5 x 10(6) M-1 s-1) and dissociation (k-1 = 2.5 s-1) enable us to estimate Ca.ATP affinity (Kd = 7 x 10(-7) M). Rapid acid quenching shows that covalent phosphoenzyme forms slowly with a rate constant of 0.6 s-1 (k2), and parallel filtration experiments indicate that phosphorylation and ADP release occur simultaneously. If the reaction is reversed (toward ATP synthesis) 70% of the phosphoenzyme is ADP-sensitive and ADP-induced dephosphorylation is fast (k-2 = 15 s-1), whereas the ADP-insensitive phosphoenzyme slowly hydrolyzes in the forward direction of the ATPase cycle at a rate of 0.05 s-1. Combination of rapid filtration and rapid acid quenching and the use of a mixture of [14C]ATP and [32P]ATP allow us to study the kinetics of several transient species. These data are used to develop a molecular mechanism for Ca-ATPase phosphorylation.

Effects of Inorganic Phosphate on the Plasma Membrane H+-ATPase from Red Beet (Beta vulgaris L.)

The effects of inorganic phosphate on the plasma membrane H(+)-ATPase of red beet (Beta vulgaris L.) were studied. ATPase activity was inhibited weakly and noncompetitively by phosphate. This anion also relieved the inhibition caused by vanadate by displacing it from the enzyme. From this effect, a dissociation constant for phosphate of 25 millimolar and an extrapolated activity at infinite phosphate concentration of 84% of the activity without inhibitors were calculated. The partial inhibition by phosphate indicates the existence of a catalytically active enzyme-phosphate complex. In the presence of 24% dimethylsulfoxide, the inhibition of ATPase activity by phosphate is much greater than in its absence. This suggests that the active enzyme-phosphate complex could be converted into a covalent phosphoenzyme through a dehydration promoted by the low water activity of the medium. The inhibitory ability of phosphate in 24% dimethylsulfoxide was dependent on the presence of potassium. Potassium ions increased both the affinity for phosphate and the inhibition caused by an infinite phosphate concentration, suggesting that potassium stimulates both phosphate binding and phosphoenzyme formation.

Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.

Stereochemistry of phospho group transfer catalyzed by a mutant alkaline phosphatase.

The stereochemical course of the phospho group transfer catalyzed by mutant (S102C) alkaline phosphatase from Escherichia coli was investigated by using 31P nuclear magnetic resonance spectroscopy. Transphosphorylation from 4-nitrophenyl (Rp)-[16O, 17O, 18O]phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This result is consistent with the view that the hydrolysis of substrates by this mutant enzyme proceeds by way of a covalent phosphoenzyme intermediate in the same manner as the wild-type alkaline phosphatase.

Phosphorylation of the calcium adenosinetriphosphatase of sarcoplasmic reticulum: rate-limiting conformational change followed by rapid phosphoryl transfer.

The calcium adenosinetriphosphatase of sarcoplasmic reticulum, preincubated with Ca2+ on the vesicle exterior (cE X Ca2), reacts with 0.3-0.5 mM Mg X ATP to form covalent phosphoenzyme (E approximately P X Ca2) with an observed rate constant of 220 s-1 (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4, 23 microM free external Ca2+, intact SR vesicles passively loaded with 20 mM Ca2+). If the phosphoryl-transfer step were rate-limiting, with kf = 220 s-1, the approach to equilibrium in the presence of ADP, to give 50% EP and kf = kr, would follow kobsd = kf + kr = 440 s-1. The reaction of cE X Ca2 with 0.8-1.2 mM ATP plus 0.25 mM ADP proceeds to 50% completion with kobsd = 270 s-1. This result shows that phosphoryl transfer from bound ATP to the enzyme is not the rate-limiting step for phosphoenzyme formation from cE X Ca2. The result is consistent with a rate-limiting conformational change of the cE X Ca2 X ATP intermediate followed by rapid (greater than or equal to 1000 s-1) phosphoryl transfer. Calcium dissociates from cE X Ca2 X ATP with kobsd = 80 s-1 and ATP dissociates with kobsd = 120 s-1 when cE X Ca2 X ATP is formed by the addition of ATP to cE X Ca2. However, when E X Ca2 X ATP is formed in the reverse direction, from the reaction of E approximately P X Ca2 and ADP, Ca2+ dissociates with kobsd = 45 s-1 and ATP dissociates with kobsd = 35 s-1. This shows that different E X Ca2 X ATP intermediates are generated in the forward and reverse directions, which are interconverted by a conformational change.

Similarities and differences between the tonoplast-type and the mitochondrial H+-ATPases of oat roots.

The native tonoplast and the mitochondrial H+-ATPase from oat roots were compared to determine whether the two enzymes have similar mechanisms. H+ pumping in low-density microsomal vesicles reflected activity from the tonoplast-type ATPase, as ATPase activity and ATP-dependent H+ pumping (quinacrine fluorescence quenching) showed similar sensitivities to inhibition by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, nitrate, quercetin, or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. The tonoplast-type ATPase was stimulated by C1-,Br- greater than HCO3- whereas the mitochondrial ATPase was stimulated by HCO3- much greater than C1-,Br-. Both enzymes hydrolyzed ATP preferentially and were inhibited competitively by AMP or ADP. Apart from resistance to azide, the tonoplast-type ATPase was strikingly similar in its inhibitor sensitivities to the mitochondrial ATPase. The insensitivity to vanadate of both enzymes suggests the reaction mechanisms do not involve a covalent phosphoenzyme. Inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide and protection by ATP suggests tyrosine and cysteine residues are in the catalytic site of the tonoplast ATPase. The mitochondrial ATPase was 100 times more sensitive to N,N'-dicyclohexyl-carbodiimide inhibition than the tonoplast H+-ATPase. These results suggest the tonoplast and the mitochondrial H+-ATPases share common steps in their catalytic and vectorial reaction mechanisms, yet sufficient differences exist to indicate they are two distinct ATPases.

Oxygen-18 isotope effect in carbon-13 nuclear magnetic resonance spectroscopy. 9. Hydrolysis of benzyl phosphate by phosphatase enzymes and in acidic aqueous solutions

The /sup 18/O isotope-induced shifts in /sup 13/C and /sup 31/P nuclear magnetic resonance (NMR) spectroscopy were used to establish the position of bond cleavage in the phosphatase-catalyzed and acid-catalyzed hydrolysis reactions of benzyl phosphate. The application of the /sup 18/O-isotope effect in NMR spectroscopy affords a continuous, nondestructive assay method for following the kinetics and position of bond cleavage in the hydrolytic process. The technique provides advantages over most discontinuous methods in which the reaction components must be isolated and converted to volatile derivatives prior to analysis. In the present study, (..cap alpha..-/sup 13/C,ester-/sup 18/O)benzyl phosphate and (ester-/sup 18/O)benzyl phosphate were synthesized for use in enzymatic and nonenzymatic studies. Hydrolysis reactions catalyzed by the alkaline phosphatase from E. coli and by the acid phosphatases isolated from human prostate and human liver were all accompanied by cleavage of the substrate phosphorus-oxygen bond consistent with previously postulated mechanisms involving covalent phosphoenzyme intermediates. An extensive study of the acid-catalyzed hydrolysis of benzyl phosphate at 75/sup 0/C revealed that the site of bond cleavage is dependent on pH. At pH less than or equal to 1.3, the hydrolysis proceeds with C-O bond cleavage; at 1.3 < pH < 2.0, there is a mixturemore » of C-O and P-O bond scission, the latter progressively predominating as the pH is raised; at pH greater than or equal to 2.0, the hydrolysis proceeds with exclusive P-O bond scission. (S)-(+)-(..cap alpha..-/sup 2/H)Benzyl phosphate was also synthesized. Hydrolysis of this chiral benzyl derivative demonstrated that the acid-catalyzed C-O bond scission of benzyl phosphate proceeds by an A-1 (S/sub N/1) mechanism with 70% racemization and 30% inversion at carbon. 37 references, 4 figures, 2 tables.« less

Stereochemical course of phospho group transfer by human prostatic acid phosphatase.

The stereochemical course of the phospho transfer catalyzed by homogeneous human prostatic acid phosphatase was investigated using 31P nuclear magnetic resonance spectroscopy. Transphosphorylation from phenyl-(R)-[15O, 17O, 18O]phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This stereochemical result is consistent with the interpretation that the hydrolysis of substrates by this enzyme proceeds by way of a covalent phosphoenzyme intermediate. Conditions for optimizing phospho transfer by this and related acid phosphatases have also been explored.

Passive rubidium fluxes mediated by Na‐K‐ATPase reconstituted into phospholipid vesicles when ATP‐ and phosphate‐free

1. Phospholipid vesicles reconstituted with Na-K-ATPase from pig kidney, show slow passive pump-mediated (86)Rb fluxes in the complete absence of ATP and phosphate.2. The Rb fluxes are inhibited in vesicles prepared from enzyme pre-treated with either ouabain or vanadate ions. Rb fluxes through Na-K pumps oriented inside-out or right-side out by comparison with the normal cellular orientation can be distinguished by effects of vanadate on one or both sides of the vesicle.3. (86)Rb uptake into Rb-loaded vesicles represents a (86)Rb-Rb exchange. The maximal rate of exchange through inside-out and right-side out oriented pumps is equal, suggesting a random arrangement of the pumps across the vesicle membrane. This Rb-Rb exchange is half-saturated on inside-out and right-side out pumps at about 0.6 and 0.2 mM-external Rb respectively.4. (86)Rb uptake into Rb-free vesicles represents a net Rb flux. The Rb uptake through inside-out pumps has a maximal rate about equal to the Rb-Rb exchange, half-saturates at an external Rb concentration of roughly 0.5 mM, and shows evidence for co-operativity. Net Rb uptake through right-side out pumps is very slow, and half-saturates at roughly 0.1 mM external Rb.5. K ions at low concentrations in the exterior medium stimulate (86)Rb uptake, but at high concentrations, inhibit. Na ions in the exterior medium always inhibit (86)Rb uptake. The result suggests that K ions are transported in co-operative fashion together with Rb ions, while Na ions block the Rb fluxes.6. The presence of Rb congeners at the vesicle interior raises the (86)Rb uptake through inside-out pumps with the decreasing order of effectiveness: Li > Na > Cs > K > Rb. Stimulation by Na ions involves a Rb-Na exchange.7. Turnover numbers were estimated from parallel measurement of Na/K pump mediated fluxes and amount of covalent phosphoenzyme. In units of moles of ion per mole of phosphoenzyme per second at 20 degrees C the following values were obtained: ATP-dependent Na-Rb exchange, 43; (ATP+phosphate)-stimulated Rb-Rb exchange, 7. For (ATP+phosphate)-independent fluxes: Rb-Rb exchange 0.25; net Rb uptake 0.15 and Rb-Na exchange 0.65.8. Mg ions in the exterior medium inhibited both net and exchange Rb fluxes through inside-out pumps in a manner antagonistic with respect to Rb. Mg and vanadate ions inhibit the Rb fluxes in a synergistic fashion.9. The results are interpreted in terms of a model in which net and exchange (86)Rb fluxes occur via conformational transitions between form E(1) which binds Rb at the cytoplasmic face of the protein, the form E(2) (Rb)(occ) containing occluded Rb ions and a form E(2) which binds Rb at the extracellular face of the protein. A kinetic analysis allows us to identify rate-limiting steps of the transport cycle by making use of our transport data in combination with values of rate-constants for conformational transitions observed directly in isolated Na-K-ATPase.

Sarcoplasmic reticulum ATPase phosphorylation from inorganic phosphate in the absence of a calcium gradient. Steady state and kinetic fluorescence studies.

The intrinsic fluorescence of sarcoplasmic reticulum vesicles was measured under conditions allowing ATPase phosphorylation from inorganic phosphate. Significant fluorescence enhancement of up to 4% resulted from gradient-independent enzyme phosphorylation at pH 6, in the absence of KCl. The equilibrium fluorescence data obtained at various magnesium and phosphate concentrations agree with a reaction scheme in which Mg2+, as direct activator, and free phosphate, as the true substrate, bind to the enzyme in random order to give a noncovalent ternary complex (Mg.*E.Pi), in equilibrium with the covalent phosphoenzyme (Mg.*E-P). The transient kinetics of the fluorescence rise was also studied, and the resulting data were generally consistent with the above scheme, assuming that binding reactions are fast compared to covalent phosphoenzyme formation. This, however, might be valid only as a first approximation. At 20 degrees C and pH 6, the phosphate concentration for half-maximum phosphorylation rate constant, at 20 mM magnesium, was higher than 20 mM. Similarly, the magnesium concentration for half-maximum phosphorylation rate constant, at 20 mM phosphate, was also higher than 20 mM. The maximum phosphorylation rate was faster than 25 s-1, and the phosphoenzyme hydrolysis rate constant was 1.5-2 s-1 under these conditions, so that the equilibrium constant between Mg.*E.Pi and Mg.*E-P largely favors the phosphoenzyme.

An essential carboxylic acid group in human prostate acid phosphatase

Treatment of homogenous human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) with low concentrations of Woodward's reagent K ( N- ethyl-5-phenylisoxazolium-3′-sulfonate ) leads to a rapid loss of enzymic activity. The rate of inactivation of the enzyme is reduced in the presence of the competitive inhibitors phosphate and l-(+)-tartrate, but not in the presence of non-inhibitory D-(−)-tartrate. Measurement of the ethylamine produced upon hydrolysis of enzyme modified in the presence of d- and of l-tartrate permitted the quantitative estimation of the number of carboxylic acid residues at the active site. The data indicate that two carboxyl groups per (dimeric) enzyme molecule are essential for catalytic activity. It is proposed that one function of the active site carboxyl group may be to protonate the leaving alcohol or phenol portion of the phosphomonoester substrate during the formation of the covalent phosphoenzyme intermediate.

An essential active-site histidine residue in human prostatic acid phosphatase Ethoxyformylation by diethyl pyrocarbonate and phosphorylation by a substrate

Human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) is a dimeric ( α 2 ) protein that catalyses the hydrolysis of phosphomonoesters. Several reports suggest that a phosphoenzyme intermediate is involved in the mechanism of acid phosphatase. Chemical modification studies and trapping experiments were therefore undertaken in order to ascertain the identity of the amino acid residue(s) involved in the formation of this intermediate. Human prostatic acid phosphatase is inactivated by diethyl pyrocarbonate (second-order rate constant of 7 M −1 · min −1 at pH 6.2) with an accompanying increase in absorbance at 242 nm due to formation of ethoxyformylhistidyl derivatives. In the presence of competive inhibitors the the rate of inactivation is decreased. Inactivation can be partially reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue having a p K a of 6.5. Direct evidence for the involvement of a histidine residue in the mechanism was obtained by trapping a covalent phosphohistidylenzyme intermediate. Incubation of the enzyme with p- nitrophenyl[ 32P]phosphate leads to incorporation of 0.44 mol 32P/mol enzyme. The denatured phosphoenzyme, which was acid labile but base stable, was hydrolyzed in 3 M KOH and the radioactivity was found to cochromatograph with synthetic τ-phosphohistidine on Dowex-1 ion-exchange resin. These results are consistent with a catalytic mechanism involving histidine as a nucleophile in the formation of a covalent phosphoenzyme intermediate.

Microdetermination of phosphorylated intermediates of sodium-potassium adenosine triphosphatase

We have described a simple, rapid, reliable, and sensitive method for measuring the covalent phosphoenzyme intermediates that form in the reaction of ATP and sodium-potassium sensitive adenosine triphosphatase. Results are reproducible even when microgram amounts of microsomal protein are used. The method involves recovery of 32P-labeled phosphoenzyme intermediates on a Millipore filter.